Platelets are critical for blood clotting. However, in various human anemias, and in bone marrow transplant patients, platelets and the megakaryocytes they are derived from can drop below a critical threshold that is required to maintain normal clotting. This can require platelet transfusions that are very expensive and which place the patient at risk for infection by blood-borne pathogens (e.g. HIV, HepB and C).
SH2-containing-5′ inositol phosphatase-1 (SHIP) can catalyze the removal of the 5′ phosphate group from PI(3,4,5)P3 (PIP3) (Damen, J. E. et al. Proc Natl Acad Sci USA, 1996, 93:1689-1693). In this manner, SHIP regulates survival and proliferation of various hematopoietic cell types. The numbers of myeloid cells and osteoclasts are increased in SHIP-deficient mice due to enhanced activity of the phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway that promotes their survival (Takeshita, S. et al. Nat. Med., 2002, 8:943-949; Helgason, C. D. et al. Genes Dev., 1998, 12:1610-1620; Liu, Q. et al. Genes Dev., 1999, 13:786-791).
Furthermore, the present inventors have shown that the number of natural killer cells are increased in SHIP-deficient mice resulting in an enhancement of engraftment of allogeneic hematopoietic stem cell grafts (Wang, J. W. et al. Science, 2002, 295:2094-2097). SHIP is also known to influence signaling pathways downstream of receptors for chemokines and cytokines involved in megakaryocytopoiesis and thrombopoiesis, such as Stromal-cell-derived-Factor 1 (SDF-1/CXCL-12) (Wang, J. F. et al. Blood, 1998, 92:756-764; Hamada, T. et al. J Exp Med., 1998, 188:539-548; Hattori, K. et al. Blood, 2001, 97:3354-3360; Avecilla, S. T. et al., Nat. Med., 2004, 10:64-71; Chemock, R. D. et al. Blood, 2001, 97:608-615), interleukin-3 (Liu, L. et al. Mol Cell Biol., 1994, 14:6926-6935), and thrombopoietin (TPO) (Lok, S. et al. Nature, 1994, 369:565-568; Drachman, J. G. et al. Proc Natl Acad Sci USA, 1997, 94:2350-2355). SHIP is phosphorylated after TPO binding to its receptor, c-mpl, leading to activation of PI3K that promotes cycling of megakaryocytes (MK) (Drachman, J. G. et al. Proc Natl Acad Sci USA, 1997, 94:2350-2355; Drachman, J. G. et al. Blood, 1997, 89:483-492; Geddis, A. E. et al. J Biol. Chem., 2001, 276:34473-34479). TPO influences MK development by controlling their proliferation, differentiation, survival and endoduplication (Kaushansky, K. et al. Nature, 1994, 369:568-571). Circulating platelets sequester free TPO, and thereby limit megakaryocytopoiesis during steady-state hematopoiesis (Kaushansky, K. N Engl J. Med., 1998, 339:746-754). Furthermore, SDF-1/CXCL12 induces transendothelial MK migration and platelet production in vitro (Wang, J. F. et al. Blood, 1998, 92:756-764; Hamada, T. et al. J Exp Med., 1998, 188:539-548) and in vivo (Hattori, K. et al. Blood, 2001, 97:3354-3360). The present inventors have also shown that it enhances human thrombocytopoiesis in xenotransplanted NOD/SCID mice (Perez, L. E. et al. Exp Hematol., 2004, 32:300-307). SHIP-deficient myeloid progenitors exhibit enhanced chemotaxis towards SDF-1/CXCL-12, indicating SHIP influences signaling downstream of CXCR-4 (Kim, C. H. et al. J Clin Invest., 1999, 104:1751-1759). In addition, SHIP has been shown to regulate PIP3 levels after thrombin or collagen activation of platelets (Giuriato, S. et al. J Biol. Chem., 1997, 272:26857-26863; Giuriato, S. et al. Biochem J., 2003, 376:199-207).
Thus, the present inventors hypothesized that SHIP may also be involved in the regulation of megakaryocytopoiesis and platelet production in vivo. It has been reported that colony forming-unit megakaryocyte (CFU-Mk) are decreased in SHIP−/− bone marrow (BM) (Moody, J. L. et al. Blood, 2004, 103:4503-10).
A naturally occurring gene-silencing mechanism triggered by double-stranded RNA (dsRNA), designated as small interfering RNA (siRNA), has emerged as a very important tool to suppress or knock down gene expression in many systems. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21-25 nucleotide fragments with characteristic 5′ and 3′ termini (Provost, P. D. et al. Embo J, 2002, 21:5864). These siRNAs act as guides for a multi-protein complex, including a PAZ/PIWI domain containing the protein Argonaute2, that cleaves the target mRNA (Hammond, S. M. et al. Science, 2001, 293:1146-1150). These gene-silencing mechanisms are highly specific and potent and can potentially induce inhibition of gene expression throughout an organism. The short interference RNA (siRNA) approach has proven effective in silencing a number of genes of different viruses (Fire, A. Trends Genet., 1999, 15:358-363).
RNA interference (RNAi) is a polynucleotide sequence-specific, post-transcriptional gene silencing mechanism effected by double-stranded RNA that results in degradation of a specific messenger RNA (mRNA), thereby reducing the expression of a desired target polypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated by double-stranded polynucleotides, such as double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is compromised. RNAi reportedly is not effected by double-stranded RNA polynucleotides that share sequence identity with intronic or promoter sequences (Elbashir et al., 2001). RNAi pathways have been best characterized in Drosophila and Caenorhabditis elegans, but “small interfering RNA” (siRNA) polynucleotides that interfere with expression of specific polynucleotides in higher eukaryotes such as mammals (including humans) have also been considered (e.g., Tuschl, 2001 Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science 296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001 Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).
According to a current non-limiting model, the RNAi pathway is initiated by ATP-dependent cleavage of long dsRNA into double-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called small interfering RNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001; Nyknen et al., Cell 107:309-21 (2001); Zamore et al., Cell 101:25-33 (2000)). In Drosophila, an enzyme known as “Dicer” cleaves the longer double-stranded RNA into siRNAs; Dicer belongs to the RNase III family of dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Further, according to this non-limiting model, the siRNA duplexes are incorporated into a protein complex, followed by ATP-dependent unwinding of the siRNA, which then generates an active RNA-induced silencing complex (RISC) (WO 01/68836). The complex recognizes and cleaves a target RNA that is complementary to the guide strand of the siRNA, thus interfering with expression of a specific protein (Hutvagner et al., supra).
In C. elegans and Drosophila, RNAi may be mediated by long double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells, however, transfection with long dsRNA polynucleotides (i.e., greater than 30 base pairs) leads to activation of a non-specific sequence response that globally blocks the initiation of protein synthesis and causes mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfection of human and other mammalian cells with double-stranded RNAs of about 18-27 nucleotide base pairs in length interferes in a sequence-specific manner with expression of particular polypeptides encoded by messenger RNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164; Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol. Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 Nature Cell Biol. 4:317).
siRNA may offer certain advantages over other polynucleotides known to the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective siRNA polynucleotide concentrations, enhanced siRNA stability, and shorter siRNA oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides). By way of a brief background, “antisense” polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S. Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; see also, e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing “dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides can be targeted to an RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. Ser. No. 2002/193579). “Triplex” DNA molecules refers to single DNA strands that bind duplex DNA to form a colinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Pat. No. 5,176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions. Because single-stranded polynucleotides do not readily diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells. siRNAs, by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polynucleotides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.
Due to its advantages, RNAi has been applied as a target validation tool in research and as a potential strategy for in vivo target validation and therapeutic product development (Novina, C. D. and Sharp, P. A., Nature, 2004, 430:161-164). In vivo gene silencing with RNAi has been reported using viral vector delivery and high-pressure, high-volume intravenous (i.v.) injection of synthetic iRNAs (Scherr, M. et al. Oligonucleotides, 2003, 13:353-363; Song, E. et al. Nature Med., 2003, 347-351). In vivo gene silencing has been reported after local direct administration (intravitreal, intranasal, and intrathecal) of siRNAs to sequestered anatomical sites in various models of disease or injury, demonstrating the potential for delivery to organs such as the eye, lungs, and central nervous system (Reich, S. J. et al. Mol. Vis., 2003, 9:210-216; Zhang, X. et al. J. Biol. Chem., 2004, 279:10677-10684; Dorn, G. et al. Nucleic Acids Res., 2004, 32, e49). Silencing of endogenous genes by systemic administration of siRNAs has also been demonstrated (Soutschek, J. et al. Nature, 2004, 432:173-178). It has been shown that siRNAs delivered systemically in a liposomal formulation can silence the disease target apolipoprotein B (ApoB) in non-human primates (Zimmermann T. S. et al., Nature, 2006, 441:111-114).